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Planning to work with aptamers?

IDT does synthesizes aptamers and aptamer libraries, and there are hundreds of published research papers describing the successful use of such sequences. Learn about aptamers, SELEX, and how IDT can assist you with reagents for your aptamer applications.

Mar 15, 2016

Revised/updated Oct 16, 2017

Aptamers (Figure 1) are short, 20–80 nt single-stranded DNA or RNA sequences or proteins that bind to target molecules with high affinity and specificity through their 3-dimensional structures. RNA sequences make up the majority of nucleic acid aptamers, perhaps because they can be synthesized by in vitro transcription in the laboratory and, with a 2′-OH, would potentially provide more diverse secondary structure than single-stranded DNA molecules. Nucleic acid aptamers are often identified using an iterative enrichment technique, such as SELEX (Figure 2), where oligos or proteins with increased affinity and specificity to a target molecule are isolated from a sequence pool after several rounds of selection.

art99-CC-aptamers-Fig1

Figure 1. Engineering aptamers to bind specific targets.

Because they have similar target binding affinities for their targets as antibodies, yet offer several advantages over antibody-based affinity molecules, aptamers are often used as substitutes for antibodies. Aptamers are typically easier to produce, especially on a large scale. They are physically more stable, and modifications that increase intracellular stability are easily incorporated, all at a lower cost. Aptamers are also easily purified and typically have low immunogenicity. They penetrate tissues to reach their target sites faster and more effectively than antibodies, due to their smaller size (8–25 kDa nucleic acids vs. ~150 kDa antibodies), and are also able to target molecules with low antigenicity; for example, when protein targets might otherwise not provide enough epitopes for antibody binding because they are fragmented or denatured [1,2].

Like antibodies, aptamers have a broad range of applications, serving as drugs, diagnostic tools, analytic reagents, bio-imaging molecules, and biosensors (aptasensors) [1–5]. Aptamers can also be used in a targeted therapeutic role by delivering nanoparticles, antibodies, and other drugs to cancer cells through conjugation to these molecules [2].

What is SELEX?

SELEX (Systematic evolution of ligands by exponential enrichment) is a recursive nucleic acid aptamer selection technology. It works by isolating sequences with increased affinity and specificity to a target molecule from an oligo sequence pool through several rounds of selection. The process proceeds as follows (also, see Figure 2):

  • A library of single-stranded DNA or RNA is generated. These sequences consist of a variable sequence region (usually 30–40 nt) flanked by static binding sites on either side.
  • The library is incubated with the selected target molecule. DNA or RNA molecules that bind to the target are prospective aptamers for that target. After unbound sequences are filtered out, the bound sequences are separated from the target and purified. Nitrocellulose membrane filtration, affinity chromatography/magnetic bead, capillary electrophoresis, microfluidic chips, atomic force microscopy (AFM), electrophoretic mobility shift assays (EMSA), and surface plasmon resonance (SPR) are some of the technologies used to separate bound from unbound sequences.
  • The bound sequences are then PCR amplified, creating a more specific sequence library. This library is used in a new round of SELEX to further optimize the quality of the aptamers.
art99-CC-aptamers-Fig2

Figure 2. Iterative selection of target specific nucleic acids using SELEX.

The SELEX procedure can fail to produce promising aptamers due to: inadequate library design, non-specific sequence retention, accumulation of amplification artifacts, and use of screening criteria that are not associated with actual application readouts [6]. With the wide range of applications for aptamers, and the limitations of SELEX, improvements and alternatives to SELEX continue to arise (e.g., High-Fidelity SELEX [6], and MAWS—Making Aptamers Without SELEX [7]).

IDT synthesizes aptamers and aptamer libraries

IDT-synthesized aptamers and aptamer libraries have contributed to hundreds of published research papers which describe the successful use of such sequences (see the Related Citations sidebar).

Most aptamers are 20–80 nt, single-stranded DNA or RNA sequences. However, IDT can synthesize longer aptamers, if needed. Base modifications can be added to aptamers for purification (e.g., 5′-biotin), for detection (e.g., 6-FAM), and to enhance stability during in vitro and in vivo use (e.g., 2′-O-Methyl RNA bases or 2′-Fluoro bases). You must provide your sequence designs, including specifying modifications and positions, for synthesis.

Base modifications increase aptamer options

Appropriate modifications and their positioning should be determined experimentally for optimal aptamer function. As an easy way to generate aptamer designs in the laboratory for initial testing, scientists will sometimes in vitro transcribe RNA aptamers. When doing this, they often include 2′-Fluoro pyrimidine base modifications to improve aptamer stability. Note, however, that these in vitro transcribed molecules will have 2′-Fluoro modifications on every pyrimidine in the sequence. RNA oligos with so many 2′‑Fluoro base insertions may not lead to the most effective aptamers. Furthermore, when the researcher turns to an oligo manufacturer to provide scaled up aptamer yields, such heavily modified aptamers can prove challenging to chemically synthesize (not to mention expensive). In fact, IDT limits standard RNA oligo synthesis to 20 or fewer 2′-Fluoro base modifications.*

How to order aptamers

To order aptamers online, visit the Custom DNA Oligos or Custom RNA Oligos ordering pages. Enter your desired scale, purification, and the sequence(s) with random bases or modifications to suit your needs.

*For aptamer sequence designs with greater than 20 2′-Fluoro bases inserted, or to include modifications not listed on our website, please submit a request for review of your design by emailing noncat@idtdna.com with your name, organization, and sequences. If you would like help with your order, contact our Customer Care.

References

  1. Song K-M, Lee S, Ban C. (2012) Aptamers and their biological applications. Sensors, 12(1):612–631.
  2. Sun H, Zhu X, et al. (2014) Oligonucleotide aptamers: New tools for targeted cancer therapy. Mol Ther Nucleic Acids, 3:e182.
  3. Brody EN, Gold L. (2000) Aptamers as therapeutic and diagnostic agents. J Biotechnol, 74:5–13.
  4. Murphy MB, Fuller ST, et al. (2003) An improved method for the in vitro evolution of aptamers and applications in protein detection and purification. Nucl Acids Res, 31(18):e110.
  5. Tombelli S, Minunni M, Mascini M. (2007) Aptamers-based assays for diagnostics, environmental and food analysis. Biomol Eng, 24(2):191–200.
  6. Ouellet E, Foley JH, et al. (2015) Hi‐Fi SELEX: A high‐fidelity digital‐PCR based therapeutic aptamer discovery platform. Biotechnol Bioeng, 112(8):1506-1522.
  7. MAWs; see http://2015.igem.org/Team:Heidelberg/Overview [Accessed 22 Feb, 2016]; and Prediger E. (2016) Functional nucleic acids as antibody alternatives for small molecule detection. Coralville, Integrated DNA Technologies. [Accessed 22 Feb, 2016].
  8. Proske D, Blank M, et al. (2005) Aptamers–basic research, drug development, and clinical applications. Appl Microbiol Biotechnol, 69(4):367–374.
  9. Keefe AD, Pai S, Ellington A. (2010) Aptamers as therapeutics. Nat Rev Drug Discov, 9(7):537–550.

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